Monitoring methods

ABSTRACT

A method of providing warning of the onset of the fertile phase of the human ovulation cycle, involving measurement in absolute or relative terms of the body fluid concentration of an analyte such as estradiol or a metabolite thereof wherein if in the current cycle a concentration measurement conducted at about the termination of menses reveals a body fluid concentration that is typical of that found in the body fluid of an average human female subject about 3 days prior to the time of ovulation during a 28-day cycle, the current cycle is immediately declared to be in its fertile phase. Where the analyte is E3G, the E3G concentration measurement is conducted on at least one or numerical days 4 to 7 of the current cycle, counting from the onset of menses, and the fertile phase is declared immediately if the E3G measurement reveals a concentration equal to or greater than a threshold concentration chosen in the range of about 25 to about 35 ng/ml.

FIELD OF THE INVENTION

[0001] This invention relates to methods of monitoring the ovulationcycle in female mammals especially humans.

BACKGROUND TO THE INVENTION

[0002] The last few decades have seen much research conducted into waysof enhancing “natural” family planning, in which physiologicalparameters indicative of the status of the ovulation cycle aremonitored. In our European patent specification EP-A-706346 weparticularly describe such a method which uses the measurement ofurinary estradiol or metabolites thereof, especiallyestrone-3-glucuronide (E3G), to provide a warning of the onset of thefertile phase. Related methods are described in our European patentspecifications EP-A-656118, EP-A-656119 and EP-A-656120. Associatedtesting devices and test kits are described in these specifications, andalso in our International patent specifications WO 95/13531 and WO96/09553. A major objective of these earlier inventions is to providemonitoring methods which are tolerant to the variability in ovulationcycle parameters that occur between different individual subjects, andindeed within the same subject from one cycle to another. Especially forcontraceptive purposes, a method should provide reliable fertilityawareness despite such variability.

[0003] Even amongst a population of individual women experiencingapparently normal-length cycles (average 28 days), some individuals mayexhibit extremely short cycle lengths, on an occasional or more frequentbasis. The whole cycle can be compressed into 20 or 21 days, or inextreme instances an even shorter interval. The fertile phase (takinginto account the time during which male sperm may remain viable) cancommence exceptionally early. In a monitoring method which is lookingfor a rise in the urinary concentration of E3G or a similar metabolite,as an indicator of imminent entry into the fertile phase, the occurenceof a very short cycle with very early commencement of the fertile phasemay not easily be identified. Accordingly, as a further refinement,there is need for a “failsafe” mechanism to cope with unexpected shortcycles.

GENERAL DESCRIPTION OF THE INVENTION

[0004] By the invention we provide a method of monitoring the humanovulation cycle, in which the cycle is immediately declared fertile if abody fluid test conducted on or about day 6 of the cycle reveals aconcentration of a fertility-related analyte which significantly differsfrom the concentration expected at that point. Taking as an exampleestradiol and its metabolites, it has hitherto been understood thatabout numerical days 5 to 7 of the cycle, counting from the onset ofmenses, the amount of estradiol and its metabolites circulating withinthe body, and hence excreted in urine and other fluids, is at or nearits lowest level within the cyclic variation, and that it is some daysthereafter before the amount rises to a level that is indicative ofimminent ovulation. Although this is the normal situation, there areexceptions.

[0005] The invention provides a method of providing warning of the onsetof the fertile phase of the human ovulation cycle, involving measurementin absolute or relative terms of the body fluid concentration of ananalyte, such as estradiol or a metabolite thereof, characterised inthat if in the current cycle a concentration measurement conducted atabout the termination of menses reveals a body fluid concentration thatis typical of that found in the body fluid of an average human femalesubject about 3 days prior to the time of ovulation during a 28-daycycle, the current cycle is immediately declared to be in its fertilephase.

[0006] A particular embodiment of the invention is a method of providingwarning of the onset of the fertile phase of the human ovulation cycle,involving measurement in absolute or relative terms of the urinaryconcentration of E3G, characterised in that the fertile phase isdeclared immediately if an E3G measurement conducted at about thetermination of menses reveals a concentration equal to or greater than athreshold concentration chosen in the range of about 25 to about 35ng/ml.

[0007] The most appropriate time when the E3G concentration measurementis conducted is on at least one of numerical days 4 to 7 of the currentcycle, and most preferably about day 6, counting from the onset ofmenses.

[0008] In a further embodiment, the invention provides a method ofmonitoring the fertility status of the mammalian ovulation cycle,involving determining a change in the body fluid concentration of ananalyte the concentration of which alters as the fertile phase of thecycle approaches and wherein a concentration measurement is made duringthe interval spanning days 4 to 8 of the current cycle, characterised inthat if the measurement reveals a concentration level at least equal tothat expected about 3 days prior to the time of ovulation, based onmeasurements taken in one or more previous cycles in the sameindividual, the onset of the fertile phase is declared immediately.Normally this method involves determining a change in the body fluidconcentration of estradiol or a metabolite thereof, such as E3G.

[0009] The invention also provides a method of monitoring the fertilitystatus of the human ovulation cycle, involving determining a change inthe urinary concentration of E3G during the early part of the cycle as awarning of the onset of the fertile phase, and wherein the E3Gconcentration is measured in relative or absolute terms on or about day6 of the current cycle, characterised in that if the E3G concentrationon or about day 6 of the current cycle is at least equal to theconcentration attained in the same individual about 3 days prior to thetime of ovulation during one or more previous cycles, the onset of thefertile phase in the current cycle is declared immediately.

[0010] For the purposes of illustration only, the invention will bedescribed in relation to the measurement of urinary analytes, andespecially “E3G” (estrone-3-glucuronide) and “LH” (luteinizing hormone).

[0011] In addition to estrone-3-glucuronide already mentioned, estradiolmetabolites that can also be assayed for the purposes of the inventioninclude estradiol-3-glucuronide, estradiol-17-glucuronide,estriol-3-glucuronide, estriol-16-glucuronide and (principally fornon-human subjects) estrone-3-sulphate. As will be appreciated from thefollowing description, the invention can readily be applied to dataderived from the measurement of body fluid concentrations of otheranalytes of significance in relation to the status of the ovulationcycle. Generally, the most suitable analytes are hormones and theirmetabolites. Follicle stimulating hormone (FSH) is an example. Examplesof alternative body fluids, which are relatively accessible, are saliva,crevicular fluid, sweat, sebum, tears and vaginal fluid. In principle,internal fluids, such as blood, can be used but are generally notpreferred because they can only be accessed by invasive techniques.

[0012] The skilled reader will also appreciate that the body fluid“concentration” of the chosen analyte or analytes need not be measuredin absolute terms, although this can of course be done if desired.Generally, it will be sufficient to assay an analyte in a manner whichyields a signal, convertible to numerical data, related to the actualconcentration, so that such data can be compared with similar dataobtained at a different stage in the cycle to determine, for example,whether or not a significant change in actual concentration hasoccurred. Accordingly, where this specification and claims refer to the“concentration” of an analyte, this expression should be interpretedbroadly.

[0013] An example of the context in which the present invention can beincorporated to advantage is a method of monitoring the fertility statusof an individual female mammalian subject, involving testing of the bodyfluid concentration of an analyte, especially estradiol or a metabolitethereof, in which method said testing is conducted at least once duringthe interval spanning days 1 to 7 inclusive of the current cycle, toestablish a reference concentration value or signal for the currentcycle, and said testing is also conducted later in the current cycle andthe concentration value or signal then obtained is compared to thereference value or signal, to detect a change indicative of imminentovulation during a normal-length cycle. The invention supplements thismethod by providing a valuable “failsafe” to detect an unusuallyshort-length cycle.

[0014] A more detailed example would be a method of monitoring thecurrent fertility status of an individual human female, involvingtesting of the body fluid concentration of estradiol or a metabolitethereof and comparing the test result with a reference value or signalto ascertain whether an elevated concentration indicative of imminentovulation is present, wherein the reference value or signal for thecurrent cycle is established by testing the body fluid concentration inthe same individual at least once during the interval spanning days 4 to7 inclusive, preferably on days 5 and/or 6, of the current cycle,testing is continued or, more preferably, recommenced on or about day 9of the current cycle and continued thereafter on at least a daily basisat least until a significantly elevated concentration is detected, andthe status of the current cycle is declared to be “fertile” for theinterval commencing on the day of significantly elevated concentrationdetection and for at least the immediately successive 12 days or untilevidence of cycle termination (e.g. commencement of menses) is obtained,whichever occurs earlier. As an optional refinement of this method, if asignificantly elevated concentration is not detected on or before day15, the cycle is declared “fertile” for the interval lasting for atleast 14, preferably 15, days immediately following day 15, or untilevidence of cycle termination is obtained, if this occurs earlier.

[0015] Adopting such procedures leads to a human contraception method,involving:

[0016] a) testing the urinary concentration of estradiol or a metabolitethereof in the female partner at least once during the interval spanningdays 4 to 7 inclusive, preferably on days 5 and/or 6, of the currentcycle to establish a reference value or signal for the current cycle;

[0017] b) testing the urinary concentration again on an at least dailybasis, preferably commencing on or about day 9 of the current cycle andcontinuing until day 15 (preferably day 14) of the current cycle; and

[0018] c) avoiding unprotected intercourse during the interval lastingfor at least 12 days immediately following the day on which asignificantly elevated urinary concentration is detected or, if asignificantly elevated urinary concentration is not detected by day 15(preferably day 14), avoiding unprotected intercourse during theinterval lasting for at least 14, preferably 15, days immediatelyfollowing day 15 (preferably day 14), in either case the intervaloptionally being terminated earlier in the event of evidence of cycletermination (e.g. commencement of menses) being obtained.

[0019] Expressed more generally, a typical method of monitoring thefertility status of an individual female mammalian subject involvestesting of the body fluid concentration of at least one analyte ofsignificance in relation to the status of the ovulation cycle during thepre-ovulation phase, wherein testing for said analyte is conducted atleast once during the interval spanning days 1 to 7 inclusive of thecurrent cycle calculated from the onset of menses (day 1 being the dayon which menstruation is first observed), to establish a referenceconcentration value or signal for said analyte in the current cycle, andthereafter testing is conducted at least once (generally repeatedly,e.g. daily) prior to a day on which ovulation is likely to occur duringthe cycle, analyte concentration values or signals obtained during saidlater or repeated testing being compared with the referenceconcentration value or signal to determine whether a concentrationchange indicative of imminent ovulation is occurring or has occurredsince the previous test.

[0020] Thus, a method of monitoring the fertility status of anindividual female subject, may involve testing of the body fluidconcentration of at least one analyte of significance in relation to thestatus of the ovulation cycle during the pre-ovulation phase, whereintesting for said analyte is conducted at least once during the intervalspanning days 1 to 7 inclusive calculated from the onset of menses (day1 being the day on which menstruation is first observed), to establish areference concentration value or signal for said analyte in the currentcycle, and then testing is conducted at least once (generallyrepeatedly, e.g. daily) during a period of days commencing at least 5,and more preferably at least 6, numerical days in advance of the meannumerical day on which actual ovulation has occurred over one or moreprevious ovulation cycles in the same individual subject, analyteconcentration values or signals obtained during said period of daysbeing compared with the reference concentration value or signal todetermine whether a concentration change indicative of imminentovulation is occurring or has occurred since the previous test.Generally, the repeated testing need not be commenced earlier than about9 days in advance of the mean ovulation day.

[0021] Preferably, the concentration reference value is established fromtest(s) conducted during the interval spanning days 4 to 7 inclusive,more preferably from test(s) conducted on day 5 and/or day 6, and mostpreferably from a single test conducted on day 6.

[0022] When it is found that the concentration is equal to or greaterthan the “failsafe” threshold value on the day that the concentrationreference value would be established, the cycle is immediately declaredto be in the fertile phase. Body fluid testing should be continued overthe next few days in order to determine actual ovulation day, or anyother parameter, which is being used to provide an indication of the endof the fertile phase.

[0023] A significant change in analyte concentration indicative ofimminent ovulation, particularly appropriate when the analyte isestradiol or a metabolite thereof, will generally be noted when theratio of the reference concentration [r] to the test concentration [i]meets the following criteria:$1.5 \leq \frac{\lbrack i\rbrack}{\lbrack r\rbrack} \leq 2.5$

[0024] In particular, especially when the analyte is E3G and thereference value is established on day 6:$\frac{\lbrack i\rbrack}{\lbrack r\rbrack} \geq 2$

[0025] If the chosen assay format by means of which concentration datais obtained yields a signal which is inversely proportional to actualconcentration, as may be the case in a competition assay, it will beappreciated by the skilled reader that the relationship between [i] and[r] signals will be the inverse of those given above.

[0026] It is generally envisaged that there can be a gap of at least oneday, and more usually several days, between establishment of theconcentration reference value and the commencement of repeated testing,during which gap no testing need be conducted. Thus, in one option, theuser performs a single test at an early stage of thee cycle, eg on day6, and several days later commences a relatively brief schedule ofrepeated, eg daily testing, which is terminated after sufficientinformation has been derived to identify the fertile phase, preferablyincluding an indication of the end of the fertile phase in that cycle.Typically this termination of testing will be on the day of LH surge, orwithin a few days thereafter, so that the remainder of the cycle istest-free.

[0027] Conveniently, the body fluid can be urine. A very suitableanalyte is therefore estradiol or a metabolite thereof, such asestrone-3-glucuronide.

[0028] The mean ovulation day can be derived for example from datacollected during at least 3, and more preferably at least 5, consecutiveprevious cycles.

[0029] Ideally, the mean ovulation day is used to calculate the testingschedule for the purposes of the current cycle, and is derived from dataobtained during at least the immediately preceding cycle.

[0030] A particularly convenient method involves the determination ofthe mean ovulation day from data obtained from a “rolling” referencebase consisting of a fixed number of consecutive cycles immediatelypreceding the current cycle. Preferably this rolling reference baseconsists of the immediately preceding 3 to 12 cycles, more preferablythe immediately preceding 5 or 6 cycles. By having such a rollingreference base, any progressive “drift” in the occurrence of ovulationin the individual concerned can be picked up and accounted for in theallocation of the next repeated testing commencement day.

[0031] To perform such methods, the user can be provided with a test kitcomprising one or more testing devices for determining the concentration(in relative or absolute terms) of said at least one analyte in saidbody fluid, together with instructions advising the user to commencesaid testing during said time interval, and means enabling a user toderive said time interval and/or a precise testing commencement day fromknowledge of the numerical day on which actual ovulation occurred duringat least one previous ovulation cycle of the user. Such a kit maycomprise a plurality of disposable body fluid testing devices, togetherwith means for reading and interpreting the results of tests performedusing said testing devices. There can be an associated replenishmentpack of disposable body fluid testing devices for use in any of themethods as set forth above, for example with directions to the user touse all of the contained disposable testing devices during the course ofa single ovulation cycle.

[0032] An advantage of such methods is that effective monitoring of theovulation cycle can be achieved using data derived solely from themeasurement of body fluid analyte concentration(s). It is unnecessary tocombine this data with other parameters. In particular, there is usuallyno need to supplement this data with routine measurement of basal bodytemperature.

[0033] By adopting a concentration reference value from data in theearly part of the current cycle, such methods avoid the need forcalibration and ensure that the base-line reference is personal to thesubject under test. This can lead to a clearer indication of thesignificant pre-ovulation concentration change, compared to previouslyproposed methods based on day-to-day measurements.

[0034] The analyte chosen for providing the warning of imminentovulation is not critical, provided that the analyte exhibits adetectable concentration change within the time interval between thecommencement of testing (as determined herein) and a safe time inadvance of actual ovulation in the current cycle, under normalcircumstances.

[0035] Although this description is provided, by way of example only, inrelation to the urinary hormones E3G, luteinizing hormone (LH), andpregnanediol-3-glucuronide (P3G), although it will be readilyappreciated that the principles of the method can be used in relation toother biochemical markers, for example the hormones estradiol andprogesterone, found for example in the blood or in saliva. The methodsdescribed herein may be used in combination with observations of otherphysiological signs of the level of fertility in a female, of which sheis aware, or can readily be made aware of, e.g. markers in other bodyfluids.

[0036] Where appropriate, ovulation day can be determined by any of theknown chemical or physiological parameters, although a preferred methodis by measuring the level of LH. Once the LH surge has been detected, itcan be said that ovulation is imminent. Also, the day of the cycle onwhich ovulation has occurred can be noted for future reference. If theLH surge is detected, and hence the day of ovulation accuratelypinpointed, it can be indicated to the user with a very high degree ofcertainty that the subject will no longer be fertile four days hence (3days after ovulation). For practical purposes, a urinary LHconcentration of 20 mIU/ml can be regarded as a universal thresholdindicative of the LH surge under virtually all circumstances.

[0037] The expression “LH surge” is used herein to mean the dramaticrise in LH concentration that precedes the event of ovulation. In theart, reference is made also to “LH max”, i.e. the peak concentration ofLH. In the majority of individuals, these are for all practical purposessimultaneous, when the cycle is monitored on a day-by-day basis.However, in a few individuals, perhaps 20% of the population, the actualpeak concentration of LH is not observed until the day following themain concentration rise. For the purposes of the invention, we prefer touse the observable rise as the critical parameter.

[0038] Alternatively, or in addition, the end of the fertile phase canbe declared on the basis of knowledge of the estradiol (or metabolitethereof) concentration, in the current cycle. Conveniently, this may bedeclared on a set day following a peak concentration value. Because thepeak concentration of urinary E3G, for example, appears to be a lessreadily detectable event than the LH surge, the E3G “peak” may bedifined by reference to a threshold value, determined for example by therelationship${\frac{\lbrack i\rbrack}{\lbrack r\rbrack} > 2.5},{{preferably} \geq 3}$

[0039] the “peak” being taken to occur on the day when this relationshipis first satisfied during the testing regime adopted in the currentcycle. The inverse relationship will apply if the E3G signal ininversely proportional to actual concentration. In some instances thismay be the same day as the significant E3G rise indicative of imminentovulation is detected. When the E3G “peak” has been detected, thefertile phase can be assumed to end on the sixth, or more safely theseventh or eighth, day later. In this embodiment, the invention providesthe option of a method of monitoring fertility in the current cyclebased solely on data derived from estradiol/metabolite assays.

[0040] Another method for predicting the end of the fertile period(though not so accurately the day of ovulation) is to measure the levelsof the urinary hormone P3G. P3G has a relatively low level in urineuntil the start of the luteal phase, at which point its level risesfairly sharply. Therefore, once an elevated level of P3G is detected, itcan be indicated to the user that the luteal phase of the cycle—ie. theterminal infertile period—has commenced. An elevated level of urinaryP3G can be based on data taken during the current and/or one or morepreceding cycles. An “elevated” P3G level can be recorded, for example,when either the level of P3G detected is greater than the sum of thefour previous recorded levels of P3G in the same menstrual cycle, orgreater than 3500 ng/ml, whichever of these two thresholds is lower andis first achieved. Once an “elevated” P3G level is recorded, the subjectcan be advised that she is infertile for the remainder of that cycle.

[0041] If desired, the detection of either LH or P3G can be used as atrigger to indicate that the subject is no longer fertile until the endof the cycle, with one hormone acting as a “back up” to the other.However, it is preferred that the detection of LH be used as a primaryindicator of whether ovulation has or is about to occur, since thedetection of LH lends itself to more accurate determination of the exactovulation day than the use of P3G.

[0042] Methods of detecting body fluid analytes, such as urinary hormonemetabolites, suitable for the purposes of this method, are well known tothose skilled in the art. In a preferred embodiment, the analyte isdetected by assay methods and devices as described in our UK patent GB2204398 and our European patent application EP-A-383619.

[0043] Where a method relies on measurement of a urine component, thismust be done on a urine sample. A variety of immunoassay techniques areavailable which enable urine components to be measured. A wide varietyof solid phase testing devices such as dipsticks and chromatographicstrips have been described in the literature, and can readily be adaptedfor use in determining urinary analytes. The device should at least becapable of indicating relative levels of analyte, eg. E3G, in thresholdbands. Examples of simple assay technology that can readily be adaptedfor use in the home is described, for example, in EP 0225054, EP0183442, EP 0186799 and GB 2204398. Disposable assay strips such asthose described in GB 2204398 which simply require to be contacted withurine and which provide an assay result in semi-qualitative form, eg. bymeans of a series of test zones on the strip which are progressivelypositive at higher urinary analyte levels, can be used. Idealstrip-format assays, for detecting both E3G and LH, are described indetail in WO 96/09553. Multiple strips that respond at different analytethresholds can be used, rather than a single strip. Alternatively, avisually readable quantitative assay can be based on progression of avisible, eg. coloured, region or “front” over a surface (eg. radialdiffusion), using for example an enzyme-labelled assay.

[0044] In a more sophisticated embodiment, a recording device can beprovided which incorporates means for reading the result of the urineassay, e.g. by measuring the absorbance by or fluorescence from an assaystrip. This may enable a more precise numerical indication to be givenof the analyte level, and further enhance the accuracy of the method. Anideal measurement system, using optical transmission, is described indetail in WO 95/13531.

[0045] In any embodiment in which two or more analytes are measuredsimultaneously, such measurement can if desired be performed using asingle body fluid testing device, eg. a device incorporating multipleassay strips, or a single strip capable of independently detecting thelevel of the different analytes.

[0046] The detailed electronics of a recording device capable ofassimilating, remembering and handling analyte concentration data, aswell as providing the preferred electronic features of the devicediscussed herein, and predicting future cycles on the basis of suchdata, can readily be provided by those skilled in the electronics artonce they have been advised of the factors that such a device must takeinto consideration, and the information that the device must provide forthe user. Such detailed electronics do not form part of the invention.However, by way of example only, reference can be made to EP-A-706346and WO 95/13531.

ESTABLISHING AN APPROPRIATE THRESHOLD

[0047] Taking E3G as an example, the following data is from a trialprogramme during which 54 women provided daily early morning urinesamples over 9 cycles. The samples were analysed for the concentrationof E3G and LH by conventional EIA assays. The mean results were asfollows: Day relative to E3G concentration ovulation (ng/ml) 0 47.5 −140.1 −2 31.3 −3 23.3 −4 18.9 −5 15.3 −6 13.6 −7 12.0 −8 10.9 −9 10.2 −109.7

[0048] The event of ovulation was taken to occur on the day following LHsurge. Day “−10” was the typical day 6 of the cycle.

[0049] It was concluded from this study that an appropriate E3Gthreshold to act as a failsafe on day 6 against the possibility of anexceptionally short cycle, was about 30 ng/ml, ie. a figure in excess ofthe early morning concentration on day −3 but lower than the earlymorning concentration on day −2. Allowing for experimental error, afigure selected within the range 25-35 ng/ml would be appropriate, theactual figure selected being a balance between the risk of failing todetect a short cycle and the desirability of avoiding an algorithm thatis over-cautious and leads to an unnecessary number of “unsafe” days ina normal cycle.

EXAMPLES

[0050] The following examples are based on information obtained during aconfidential trial conducted in the UK in 1996, during which 625volunteer couples used a urinary hormone testing kit to determine thefertility status of the ovulation cycle, and relied on this fertilityawareness as their sole means of contraception. The urinary analyteswere E3G and LH, measured using test sticks as described in WO 95/13531.In accordance with the principles set forth in EP-A-706346 a baselineconcentration for E3G was established on day 6 of each cycle, and asignificant rise in E3G used as warning of the onset of the fertilephase; this was supplemented by a calendar calculation from which thefertile phase was automatically declared 3 days in advance of the mostlikely LH surge day based on previous cycles. Pregnancies resulted in anumber of instances. At the completion of the trial the electronicmonitors were returned and their memories downloaded into a computer sothat the trial histories could be evaluated and reasons for the unwantedpregnancies identified.

[0051] In accordance with WO 96/09553, the measurement of urinary E3Gconcentration was conducted using a competition assay format. The signalgenerated by the assay device (percentage of optical transmissionthrough the relevant zone of the test strip) was inversely proportionalto the actual concentration of E3G in the urine sample. The E3G assaywas standardised using highly purified crystalline estrone-3-glucuronide(E₁-3-G) supplied by Sigma Chemical Co (product code E 1752), dissolvedinto 0.01M phosphate buffer with 0.85% (w/v) saline and 0.1% (w/v)sodium azide.

[0052] On analysis of the trial date it became clear that severalpregnancies occured because the relevant cycle had been very muchshorter than anticipated and that the fertile phase had actually beenentered before the algorithm actually declared it.

[0053] The following three examples give the relevant trial data forvolunteers who became pregnant during the course of the trial and wherethe pregnancy was found retrospectively to be the result of anexceptionally short cycle length. In each instance it can be seen thatif there had been an overriding requirement that there be an immediatedeclaration of the fertile phase if an E3G signal of less than 20%transmission had been recorded on day 6, these pregnancies could havebeen avoided.

[0054] In the test kit as used for the purposes of this trial, a 20%transmission level for the E3G assay was equivalent to an E3G bufferconcentration of 30±5 ng/ml. According to these trial results, a“failsafe” E3G concentration chosen within the range 25 to 35 ng/ml andapplied on or about day 6 of the cycle would provide a safeguard againstthe consequences of an unexpectedly short cycle length.

Example 1

[0055] Cycle Length LH Surge Day 1 26 14 2 25 x 3 26 14 4 24 x 5 25 14 627 13 7 32 x 8 26 13 9 P 11 Pregnancy Cycle

[0056] Cycle 9 is the pregnancy cycle and the LH surge is the earliestin the observed sequence.

[0057] The test signals for this volunteer in the pregnancy cycle were:Day  6  9 10 11 12 13 14 15 LH  2  2  5 15  5  4  3  5 E3G 18 16 11 1415 21 15 16

[0058] In this cycle, the start of the fertile phase was declared on day10. Unfortunately, this allowed the volunteer to have unprotectedintercourse in the apparently “safe” period on days 7, 8 and 9. Thevolunteer conceived in this cycle, and this was most likely as a resultof one of these acts.

[0059] Imposing a threshold of 20% T on day 6 would have resulted in thesystem declaring the start of the fertile phase immediately.

Example 2

[0060] Cycle Length LH Surge Day 1 31 17 2 28 x 3 28 14 4 30 17 5 29 176 29 15 7 31 17 8 P 14

[0061] The pregnancy cycle has the equal earliest LH surge of allpreviously observed and is a shorter cycle than normal for thisindividual.

[0062] The test signal profile in the pregnancy cycle is: Day  6 11 1213 14 15 16 17 LH  4  6  6  5 17  9  7  6 E3G 20 16 12 14 13 14 16 18

[0063] The start of fertile phase was declared on day 12.

[0064] There was a single act of intercourse on day 9, before the LHsurge (together with acts on days 3,4,5 but these were too far away fromthe LH surge for sperm to survive, according to accepted scientificknowledge).

[0065] By imposing a day 6 20% T threshold, the fertile phase would havebeen declared immediately, thus advising the user against the mostlikely act on day 9.

Example 3

[0066] Cycle Length LH surge Day 1 30 16 2 24 x 3 24 12 4 26 13 5 p 11Pregnancy Cycle

[0067] Again the LH surge is at the earliest point in the observedsequence.

[0068] The signal profile in the pregnancy cycle is: Day  6  8  9 10 1112 13 14 LH  4  1  0  6 13  9  5  0 E3G 15 25 20 13 10 14 20 18

[0069] The declared start of the fertile phase was on day 10. Acts ofintercourse were reported on days 4, 6, 7, 8 and 9, all in theapparently “safe” phase.

[0070] By imposing a day 6 absolute threshold of 20% T, the start of thefertile phase would have been declared on day 6, thus advising againstintercourse on days 6, 7, 8 and 9. The day 4 act was too far from the LHsurge day, and hence from ovulation, for sperm to survive.

1. In a method of providing warning of the onset of the fertile phase ofthe human ovulation cycle, involving measurement in absolute or relativeterms of the body fluid concentration of an analyte indicative offertile status, the improvement that if in the current cycle aconcentration measurement conducted at about the termination of mensesreveals a body fluid concentration that is typical of that found in thebody fluid of an average human female subject about 3 days prior to thetime of ovulation during a 28-day cycle, the current cycle isimmediately declared to be in its fertile phase.
 2. An improved methodaccording to claim 1, wherein said analyte is selected from the groupconsisting of estradiol and metabolites thereof.
 3. An improved methodaccording to claim 1, wherein the urinary concentration ofestrone-3-glucuronide (E3G) is measured.
 4. In a method of providingwarning of the onset of the fertile phase of the human ovulation cycle,involving measurement in absolute or relative terms of the urinaryconcentration of E3G, the improvement that the fertile phase is declaredimmediately if an E3G measurement conducted at about the termination ofmenses reveals a concentration equal to or greater than a thresholdconcentration chosen in the range of about 25 to about 35 ng/ml.
 5. Animproved method according to claim 4, wherein said E3G concentrationmeasurement is conducted on at least one of numerical days 4 to 7 of thecurrent cycle, counting from the onset of menses.
 6. In a method ofmonitoring the fertility status of the mammalian ovulation cycle,involving determining a change in the body fluid concentration of ananalyte the concentration of which alters as the fertile phase of thecycle approaches and wherein a concentration measurement is made duringthe interval spanning days 4 to 8 of the current cycle, the improvementthat if the measurement reveals a concentration level at least equal tothat expected about 3 days prior to the time of ovulation, based onmeasurements taken in one or more previous cycles in the sameindividual, the onset of the fertile phase is declared immediately. 7.An improved method according to claim 6 applied to the human ovulationcycle, which method involves determining a change in the body fluidconcentration of an analyte selected from the group consisting ofestradiol and metabolite thereof.
 8. An improved method according toclaim 7, wherein said analyte is E3G.
 9. In a method of monitoring thefertility status of the human ovulation cycle, involving determining achange in the urinary concentration of E3G during the early part of thecycle as a warning of the onset of the fertile phase, and wherein theE3G concentration is measured in relative or absolute terms on or aboutday 6 of the current cycle, the improvement that if the E3Gconcentration on or about day 6 of the current cycle is at least equalto the concentration attained in the same individual about 3 days priorto the time of ovulation during one or more previous cycles, the onsetof the fertile phase in the current cycle is declared immediately.